U.S. patent application number 10/173586 was filed with the patent office on 2003-01-09 for novel and practical serological assay for the clinical diagnosis of leishmaniasis.
Invention is credited to Martin, Samuel K., Ryan, Jeffrey R., Smithyman, Anthony M..
Application Number | 20030008332 10/173586 |
Document ID | / |
Family ID | 22610939 |
Filed Date | 2003-01-09 |
United States Patent
Application |
20030008332 |
Kind Code |
A1 |
Ryan, Jeffrey R. ; et
al. |
January 9, 2003 |
Novel and practical serological assay for the clinical diagnosis of
leishmaniasis
Abstract
Methods for the diagnosis of visceral, cutaneous and canine
leishmaniasis in a subject suspected of being infected with the
parasitic protozoa Leishmania is disclosed. Disclosed are
antibody-capture enzyme-linked immunosorbent assays (ELISAs) for
the detection of antibodies to Leishmania parasite soluble antigens
and antigen-capture ELISAs for the detection of Leishmania parasite
soluble antigens in host samples. Also disclosed are
immunodiagnostic kits for the detection of Leishmania parasite
circulating antigens or IgM and IgG antibodies in a sample from
subject having visceral, cutaneous or canine leishmaniasis. In
these methods and kits, detection may be done photometrically or
visually. The methods and kits also allow the visualization of
Leishmania amastigotes or promastigotes in a sample.
Inventors: |
Ryan, Jeffrey R.; (Reston,
VA) ; Martin, Samuel K.; (Burtonsville, MD) ;
Smithyman, Anthony M.; (Collaroy, AU) |
Correspondence
Address: |
Office of the Staff Judge Advocate
U.S. Army Medical Research and Materiel Command
ATTN: MCMR-JA (Ms. Elizabeth Arwine)
504 Scott Street
Fort Detrick
MD
21702-5012
US
|
Family ID: |
22610939 |
Appl. No.: |
10/173586 |
Filed: |
June 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10173586 |
Jun 18, 2002 |
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09725182 |
Nov 29, 2000 |
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60168300 |
Dec 1, 1999 |
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Current U.S.
Class: |
435/7.22 |
Current CPC
Class: |
G01N 33/56905 20130101;
Y02A 50/30 20180101; C07K 14/44 20130101; G01N 2469/20
20130101 |
Class at
Publication: |
435/7.22 |
International
Class: |
G01N 033/53; G01N
033/569 |
Goverment Interests
[0002] This invention was made by employees of the United States
Army. The government has rights in the invention.
Claims
1. An immunoassay for detecting exposure to Leishmania parasites in
a subject comprising the steps of: contacting a sample from the
subject suspected of having leishmaniasis with a soluble antigen
prepared by utilizing a protein-free medium; and detecting the
presence or measuring the amount of an antibody or fragment thereof
in the sample bound to the soluble antigen.
2. The immunoassay of claim 1 wherein the protein-free medium
comprises D, xylose.
3. The immunoassay of claim 1 wherein the protein-free medium
further comprises at least one of the following ingredients: Hepes
buffer, L-glutamine and sodium bicarbonate without phenol red.
4. The immunoassay of claim 1 wherein the antibody is IgG or IgM
and is specific for a Leishmania antigen.
5. The immunoassay of claim 1 wherein the sample is a serum
sample.
6. The immunoassay of claim 5 wherein the serum sample is modified
by diluting it 1:1000 in blocking buffer having 1.0% boiled
casein.
7. The immunoassay of claim 1 wherein said immunoassay is capable
of diagnosing visceral, cutaneous or canine leishmaniasis in a
subject.
8. The immunoassay of claim 1 wherein the Leishmania soluble
antigen preparation is prepared by using clones of Leishmania
donovani or Leishmania mexicana.
9. An immunoassay for diagnosing leishmaniasis in a subject
comprising the steps of: contacting a sample from the subject with
an antibody or fragment thereof that specifically binds the
Leishmania exo-antigen; and detecting the presence or measuring the
amount of said antibody or fragment thereof bound to said
Leishmania exo-antigen.
10. The immunoassay of claim 9 wherein the antibody or fragment
thereof is adsorbed onto a substrate.
11. A kit for the diagnosis of leishmaniasis in a subject
comprising a substrate and a soluble antigen of either L. donovani
or L. mexicana prepared by utilizing a protein-free medium packaged
together for multiple or single use assays.
12. The kit of claim 11 wherein the substrate is coated with the
soluble antigen.
13. The kit of claim 11 further comprising a positive control.
14. The kit of claim 11 further comprising a negative control.
15. The kit of claim 11 further comprising a diluent.
16. The kit of claim 11 further comprising an anti-human IgG
conjugated to a label.
17. The kit of claim 11 further comprising a substrate
chromogen.
18. The kit of claim 11 further comprising a substrate buffer.
19. The kit of claim 11 further comprising a blocking buffer.
20. The kit of claim 11 further comprising a stopping solution.
21. A kit for the detection of an exo-antigen of a leishmania
parasite in a sample comprising a fluorescein-labeled antibody that
binds the exo-antigen and a counter-stain packaged together for
multiple or single use assays.
22. The kit of claim 21 further comprising a protein stabilized
buffer solution.
23. The kit of claim 21 further comprising sodium azide.
24. The kit of claim 22 wherein the fluorescein-labeled antibody is
diluted in the protein stabilized buffer solution.
25. The kit of claim 21 wherein the counter-stain is Evans
Blue.
26. The kit of claim 21 further comprising a solid substrate to
which the sample to be tested is fixed.
27. The kit of claim 11 further comprising instructions.
28. The kit of claim 21 further comprising instructions.
29. A diagnostic device comprising a Leishmania soluble antigen
prepared by utilizing a protein-free medium and a means for
detecting an antibody bound to the Leishmania soluble antigen.
30. A diagnostic device comprising an antibody or fragment thereof
that binds an exo-antigen found in a conditioned medium made by
cultivating a Leishmania parasite in a protein-free medium.
31. A method of preparing a diagnostic device comprising adsorbing
to a substrate an antibody or fragment thereof which binds an
exo-antigen found in conditioned medium made by cultivating a
Leishmania parasite in a protein-free medium.
32. A method of preparing a diagnostic device comprising adsorbing
to a substrate a soluble antigen of a Leishmania parasite prepared
by utilizing a protein-free medium.
33. A method of detecting a Leishmania parasite in a sample
comprising contacting the sample with an antibody specific for an
exo-antigen found in a conditioned medium made by cultivating the
Leishmania parasite in a protein-free medium; and detecting the
presence or measuring the amount of an antibody or fragment thereof
in the sample bound to an antigen in the sample.
34. A protein-free medium comprising an agent that balances the
oncotic pressure across a semi-permeable cell membrane.
35. The protein-free medium of claim 34, wherein the agent is D,
xylose.
36. The protein-free medium of claim 34, further comprising at
least one of the following ingredients: Hepes buffer, L-glutamine
and sodium bicarbonate without phenol red.
37. A method for obtaining an exo-antigen from an organism
comprising culturing the organism in a protein-free medium.
38. The method of claim 37, wherein the organism is a leishmania
parasite.
39. The method of claim 37, wherein the protein-free medium is
XOM.
40. The exo-antigen obtained according to the method of claim 37.
Description
RELATED APPLICATION DATA
[0001] This application claims the benefit of U.S. Provisional
Patent Application No. 60/168,300, filed Dec. 1, 1999, naming
Jeffrey R. Ryan, Samuel K. Martin, and Anthony M. Smithyman as
co-inventors, which is herein incorporated by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention relates generally to a method of diagnosing
leishmaniasis in a subject suspected of being infected with the
parasitic protozoa Leishmania. In particular, the invention relates
to enzyme-linked immunosorbent assays (ELISAs) for the detection of
Leishmania parasite circulating antigens and Leishmania-specific
antibodies in host samples.
[0005] 2. Description of the Related Art
[0006] Leishmaniasis is a serious and sometimes fatal disease
estimated by the World Health Organization (WHO) to affect
approximately 12 million people in 88 countries. Recent epidemics
of leishmaniasis have occurred in the Africa, the Indian
subcontinent and Brazil. In addition to the concerns regarding
significant morbidity and mortality caused by leishmaniasis in
endemic areas, the increase in global travel concomitant with the
increased incidence of the disease in HIV-infected and intravenous
drug-user populations is a cause for concern in non-endemic areas
more recently.
[0007] Unfortunately, current acceptable diagnostic practices lack
the means for efficiently and accurately identifying those infected
or exposed to the disease-causing parasite as explained in Martin,
S. et al. (1998) Military Medicine 163(23):801-807. As a result,
the prevention of leishmanial epidemics is greatly hindered and
patient management is difficult. Additionally, there is an imminent
threat of a developing population of people co-infected with HIV
and Leishmania. To date, there are no antigen-detection type
diagnostic tests available for Leishmaniasis. In view of this,
antigen-detection assays are desperately needed for diagnosis,
patient management and epidemiological studies.
[0008] Recombinant kinesin protein, rK39, is one of the few
antigens that has been used in the development of
antibody-detection immunoassays for active visceral leishmaniasis
(VL). However, assays of this antigen and others fail to
consistently detect antibodies in other clinical syndromes
associated with a predominately T-cell and muted B-cell response.
Moreover, antibody-detection assays have an inherent
limitation--dependence on the immune response of the host to the
parasite antigen. For example, in an immunocompromised host, the
infection may not produce proportional antibody production and
thereby escape detection.
[0009] Generally, the serological tests for diagnosing VL are
highly sensitive (>90%). See Senaldi, G., et al., (1996) J.
Immunol. Methods 193:9-5. These serological tests, however, pose
problems of non-specificity resulting in false positive results
from reference samples of other infectious diseases and subclinical
leishmanial infections. Modifications of the antigens used for
direct agglutination test (DAT) and for ELISA are reported to be
markedly successful in eliminating false-positive results. See
Zijlstra, E. E., et al., (1997) Trans. R. Soc. Trop. Med. Hyg.
91:671-673 and Badaro, R., et al., (1996) J. Inf. Dis. 173:758-761.
Unfortunately, serological tests are rarely performed to diagnose
cutaneous leishmaniasis because the sensitivities and specificities
are disappointingly poor for that clinical manifestation. See
Sanchez, J. L., et al., (1992) Am. J. Trop. Med. Hyg. 47:47-54;
Garcia-Miss, M. R., et al., (1990) Trans. R. Soc. Trop. Med. Hg.
84:356-358.
[0010] The antigens used in immunoassays for the detection of
leishmaniasis are traditionally derived from promastigotes
cultivated in vitro, or from recombinant proteins. See Badaro et
al. (1996); Choudhary, S., et al., (1992) J. Comm. Dis. 24:32-36;
Badaro, R., et al., (1986) Am. J. Trop. Med. Hyg. 35:72-78;
Choudhary, A., et al., (1990) Trans. R. Soc. Trop. Med. Hyg.
84:363-366; and Reed, S. G., et al., (1990) Am. J. Trop. Med. Hyg.
43:632-639. The promastigotes release metabolic products into the
culture medium to produce conditioned medium. These metabolic
products are immunogenic to the host. See Schnur, L. F., et al.,
(1972) Isrl. J. Med. Sci. 8:932-942; Sergeiev, V. P., et al.,
(1969) Med. Parasitol. 38:208-212; El-On, J., et al., (1979) Exper.
Parasitol. 47:254-269; and Bray, R. S., et al., (1966) Trans. R.
Soc. Trop. Med. Hyg. 60:605-609.
[0011] The prior art assays are based on these metabolic products
found in the in vitro culture medium. Thus, the presence of complex
proteins or serum components required for growth of the parasites
in culture pose several problems in the prior art assays. For
example, the amount of manipulation needed to purify the targeted
antigens from the metabolic products can affect the native
composition of certain components necessary for a highly sensitive
assay. Furthermore, insufficient purification techniques may create
problems with specificity as common proteins remain in the antigen
preparations, which cause non-specific reactions. Generally, the
prior art assays are limited in scope to one species complex or
clinical manifestation and have never demonstrated combined
sensitivity and specificity of more than 90%.
[0012] Thus, a need exists for highly sensitive and highly specific
assays for detecting, measuring, or diagnosing exposure to
Leishmania.
SUMMARY OF THE INVENTION
[0013] The invention relates to an immunoassay for detecting IgM
and IgG antibodies in a sample from a subject having visceral,
cutaneous or canine leishmaniasis.
[0014] The invention also relates to an immunoassay for detecting
Leishmania parasite circulating antigens in a sample from a subject
having visceral, cutaneous or canine leishmaniasis.
[0015] In one embodiment, the invention relates to a leishmaniasis
immunoassay, which is based on soluble antigens from promastigotes
cultivated in a protein-free and serum-free medium.
[0016] In another embodiment, the invention relates to the use of
protein-free and serum-free medium to cultivate the promastigotes
in an immunoassay for the diagnosis of leishmaniasis.
[0017] In a preferred embodiment, the assay of the invention has a
high degree of sensitivity and specificity. For example, the assay
of the invention has a combined sensitivity and specificity of more
than about 90%. Preferably, the assay of the present invention has
sensitivity of about 95.1% and a specificity of about 100%.
[0018] The invention also relates to an antigen-capture immunoassay
for the detection of exo-antigens released by the parasite in a
sample from subject having visceral, cutaneous or canine
leishmaniasis.
[0019] The invention also relates to immunodiagnostic kits for the
detection of specific IgM and IgG antibodies to Leishmania parasite
circulating antigens in a sample from a subject having visceral,
cutaneous or canine leishmaniasis. Detection may be done
photometrically or visually. If visual detection is desired, a
fluorescent chromogenic or chemiluminescent agent may be utilized.
Preferably, the intensity or amount of the visual signal is in
proportion to the amount of the antibody present in the sample.
[0020] In one embodiment, the invention allows the visualization of
Leishmania amastigotes or promastigotes by the use of a fluorescein
conjugated polyclonal antibody to the specific antigen preparation
aforementioned.
[0021] Preferably, the protein-free and serum-free medium utilized
in the present invention comprises a cross-linking agent that is
not metabolized by cultures and is relatively easy to purify out by
dialysis. In a preferred embodiment of the invention the medium
comprises xylose as xylose is not metabolized by cultures.
Additionally, only small amounts of xylose are required. Since only
small amounts of xylose are necessary, purification is relatively
easy and less destructive to the components found in the
conditioned medium.
DESCRIPTION OF THE DRAWINGS
[0022] This invention is further understood by reference to the
drawings wherein:
[0023] FIG. 1 is a boxplot to illustrate specific IgG antibody
levels measured in VL patient sera samples.
[0024] FIG. 2 is a boxplot showing specific IgM antibody levels
measured in the same samples.
[0025] FIG. 3 is a boxplot illustrating specific IgG antibody
levels measured in CL patient sera samples.
[0026] FIG. 4 is a boxplot depicting specific IgM antibody levels
measured in the same samples.
[0027] FIG. 5 shows specific IgG antibody levels measured in canine
leishmaniasis sera samples.
[0028] FIG. 6 is a boxplot that displays specific IgM antibody
levels measured in the same samples.
[0029] FIG. 7 is a Western Blot illustrating the striking rabbit
pattern (lanes 1 and 2) similar to that of the kala azar patients
pattern (lanes 5 and 6).
[0030] FIG. 8 is a graph illustrating the specific activity of the
competitive Ag-capture ELISA while using different antigen and
HRP-labeled antigen conditions.
[0031] FIG. 9 is a graph illustrating the specific activity of the
competitive Ag-capture ELISA while titrating labeled antigen under
different coating and blocking conditions.
DETAILED DESCRIPTION OF THE INVENTION
[0032] Recently, the soluble antigens shed, excreted and secreted
by Leishmania parasites in a protein-free medium have been
described. The soluble antigens of L. donovani promastigotes, are
primarily lipophosphoglycan (LPG), and comprise an albumin binding
site, a hydrophylic LPG component, and a repeating phosphorylated
saccharide linked with secreted acid phosphatase (S-AcP). See
Greis, K. D., et al., (1992) J. Biol. Chem. 267:5876-5881. S-AcP is
presumably the most immunogenic of all the glycoproteins and is a
major component in L. donovani promastigote conditioned medium. See
Bates, P. A., et al., (1988) Exper. Parasitol. 67:199-209.
Monoclonal antibody (MAb) studies show no cross reactivity between
S-AcP and parasite surface membrane or host acid phosphatases. See
Bates, P. A., et al., (1987) Exper. Parasitol. 64:157-164. S-AcP
from L. donovani promastigote conditioned medium has been used to
immunoprecipitate specific antibody from pooled sera of patients
acutely ill with VL (kala-azar). See Ellis, S. L., et al., (1998)
Exp. Parasitol. 89:161-168.
[0033] The use of a soluble antigen preparation from Leishmania
donovani to capture specific IgG antibodies in the sera of
kala-azar patients indicates that the soluble antigens found in
conditioned medium can act as the foundation for leishmaniasis
immunoassays. See Martin et al. (1998) An. Trop. Med. &
Parasit. 92: 571-577.
[0034] Thus, the present invention generally relates to
leishmaniasis immunoassays, which are highly sensitive and specific
and allow the detection of specific IgG and IgM antibodies in
subjects affected with visceral, cutaneous, or canine
leishmaniasis. The present invention also relates to
antigen-capture immunoassays which allow the detection of the
soluble Leishmania antigens.
[0035] The present invention relates to an immunoassay that allows
the detection of IgM and IgG antibodies in subjects affected with
visceral, cutaneous, or canine leishmaniasis. The assay is based on
soluble antigens from promastigotes cultivated in a protein-free
and serum-free medium and takes less than four hours to
perform.
[0036] Prior research attempts employing non-recombinant antigens
in the design of serologic tests for leishmaniasis have been
limited by problems with sensitivity, specificity and test
reproducibility. Reasons for these limitations remain elusive, but
are most likely attributable to physical and chemical techniques
used in antigen preparation. Therefore, the present invention
relates to sensitizing plates with soluble antigens from
conditioned media. Plate sensitization ameliorates the prior art
problems associated with physical and chemical techniques used in
antigen preparation.
[0037] In order to obtain soluble antigens from conditioned media,
cells must be maintained within defined physiological ranges to
thrive in vitro. These conditions include temperature, pH, osmotic
pressure, O.sub.2 and CO.sub.2 gas tension, and nutrients. It has
been shown that cells fail to thrive in culture media unless 10 to
20% serum albumin is added and the viability and growth rates are
compromised where serum albumin concentrations vary substantially
from 10%. It is believed that the primary role of serum albumin in
in vivo and in vitro cell culture systems is to balance oncotic
pressure across the semi-permeable membrane of cells and to provide
free water homeostasis. It is also believed that the critical
component of serum for in vitro cell survival is albumin.
[0038] Therefore, one aspect of the present invention is the
propagation of the promastigotes in serum-free and protein-free
medium comprising an agent that balances the oncotic pressure
across the semi-permeable membrane of the cells. An example of this
agent is xylose. Thus, the uncharacterized soluble factors in
conditioned medium from the cultivation of promastigotes in
protein-free and serum-free medium can be used as a foundation for
an immunoassay for visceral, cutaneous, or canine leishmaniasis.
The use of a defined protein-free and serum-free culture medium
reduces antigen production to a simple and inexpensive
centrifugation step and greatly increases the sensitivity,
specificity, reproducibility and practicability of the assay.
Additionally, gram quantities of the soluble antigens may be
inexpensively and reproducibly generated by methods standard in the
art. Furthermore, reactivity to the soluble antigens appears to be
genus specific.
[0039] Since sensitivity to oncotic imbalance varies with cell
type, one may wish to conduct experiments to determine the optimum
concentration range of the agent which balances the oncotic
pressure across the semi-permeable membrane of a given cell type.
For example, parasites may be cultured in a serum-free and
protein-free medium, wherein a colloidal agent balances the oncotic
pressure. The growth index may be determined by comparing the
growth of the parasites in the serum-free and protein-free medium
with the growth of the parasites in serum supplemented media. Based
on the comparison, one may then choose the optimum concentration
range of the colloidal agent.
[0040] When attempting to detect specific IgM, normally one must
consider the effect of IgG in the sample and compensate for its
presence or eliminate it altogether. Thus, a fraction of samples
tested (n=100) were subjected to a Rapi-Sep spin column (INDX),
which binds the IgG in the samples to the membranes, before testing
for specific IgM. There was no significant change in the IgM values
derived for any sample.
[0041] In preliminary studies, 129 visceral (Brazil, Italy, North
Africa, Nepal) and 143 cutaneous (Brazil) leishmaniasis patients
with controls were tested. These studies show an overall
sensitivity of 95.1% when Leishmania-specific IgG was measured
against that of healthy, North American negative controls. No
cross-reactivity was noted when this assay was used to look for
cross-reacting antibodies in patient samples from other parasitic
diseases (malaria, echinococcosis, Africa trypanosomiasis, and
filariasis). See Martin et al.
[0042] In additional studies, the assay of the present invention
correctly identified 42 sera from Brazilian dogs with canine
leishmaniasis and 10 healthy controls. As shown in FIGS. 1-6, the
difference between negative and positive was greater in the case of
dog sera than of human sera. The negative control sera subset gave
a negative cutoff score of about 0.225 for the IgG assay. FIG. 1 is
a boxplot to illustrate specific IgG antibody levels measured in VL
patient sera samples. With respect to specific IgG, all 129
clinically confirmed positive VL patient sera gave OD readings
above the negative cutoff (100% sensitivity). FIG. 2 is a boxplot
showing specific IgM antibody levels measured in the same samples.
When measuring IgM, the negative control sera tested gave a
negative cutoff score of about 0.310. The sensitivity for this
assay was 94.57% (122/129 positive).
[0043] Currently, the assay is able to detect specific IgG and IgM
antibodies with varying degrees of success in patient serum samples
from known-positive cases of visceral leishmaniasis (VL) and
cutaneous leishmaniasis (CL). The assay may be improved by
modifying serum sample and conjugate dilution, substrate,
incubation times, temperatures, and other assay conditions, such as
described in Example 8, and is well within the skill and knowledge
of one of ordinary skill in the art. For example, the serum may be
diluted by 1:1000 instead of 1:200 and all incubations may be at
room temperature instead of a 37.degree. C. humidity chamber. Serum
samples may be diluted from about 1:250 to about 1:1,000.
Preferably, for CL assays, the serum sample is diluted to 1:250 and
for VL assays, the serum sample is diluted to 1:1,000. The wide
range of conjugates that may be produced from polyclonal antibodies
against these antigens vary in their affinity and avidity. As such,
their working dilutions range from 1:5,000 to about 1:32,000.
However, one of ordinary skill in the art may readily determine the
optimum dilutions for a given sample and assay by methods standard
in the art such as checkerboard titrations.
[0044] Initially, all categories of patient samples were tested
with the WR0130E L. donovani antigen (ATCC strain 30503). However,
the assay was not sensitive to CL patient sera samples. Thus, these
CL samples were retested with the L. mexicana (ATCC strain 50157)
antigen.
[0045] The immunoassay of the present invention may detect IgM and
IgG antibodies in human patients with visceral and cutaneous
leishmaniasis, and dogs with canine leishmaniasis. When using
Leishmania-specific IgG antibodies as a marker for active disease
the test showed an overall sensitivity of 95.9% (261/272) and a
specificity of 100%.
[0046] The following examples are intended to illustrate but not to
limit the invention.
EXAMPLE 1
Sera Preparation
[0047] Serum was collected from human patients who were admitted to
clinics in Brazil, Italy, North Africa, Nepal and Walter Reed Army
Medical Center and who had either splenic aspirates or skin
biopsies from lesions that tested positive for Leishmania parasites
by culture and microscopy. In total, 129 visceral (Italy, Brazil,
North Africa, and Nepal) and 143 cutaneous (Brazil) leishmaniasis
patients (136 - L. braziliensis-infected, and 7 L.
panamensis-infected) with controls were tested.
[0048] Human negative controls were from 12 non-endemic area normal
patients with no documented infection or exposure to Leishmania
parasites. In addition to the human manifestations assayed, sera
from 42 Brazilian dogs with a clinical diagnosis for canine
leishmaniasis were tested against positive control sera from a
commercial source (Bordier Affinity Products, S. A., Crissier,
Switzerland) and 10 negative controls from a pathogen-free, canine
research colony (College of Veterinary Medicine, North Carolina
State University, Raleigh, N.C., USA).
EXAMPLE 2
Antigen Preparation
[0049] The Leishmania soluble antigen (exo-antigen) preparation was
made by cultivating Leishmania promastigotes in normal supplemented
media (RPMI, MEM plus FBS) at 26.degree. C. until the culture
reached mid-log phase at a density of about 10.sup.9 cells/ml. Then
the cells were pelleted and washed 6 times in a defined,
protein-free medium such as XOM available from GIBCO BRL, formula
number 96-0051DJ, RPMI Medium 1640 comprising D, xylose at 0.076
mM, Hepes buffer at 25 mM, L-glutamine, and sodium bicarbonate at
30 mM without phenol red.
[0050] The cells were then resuspended in protein-free medium such
as XOM to a final density of 10.sup.8 promastigotes/ml and
incubated at 26.degree. C. in a roller bottle with 0.01% Tween 80
(Sigma Chemical Co., St. Louis, Mo.) for 72 hours. The cells were
pelleted by centrifugation at 9,000.times. g for 30 minutes and the
supernatant was collected. The relative protein concentration of
the soluble antigens was estimated by measuring the optical density
at 280 nm. The antigen may be stored at 4.degree. C.
EXAMPLE 3
Antibody and Conjugate Production
[0051] The leishmania soluble antigen preparation produced by the
method explained in Example 2 was used without an adjuvant to
immunize rabbits. The antiserum was pooled and affinity-purified on
a Protein A column containing the antigen preparation of Example 2
above. Fractions of the polyclonal antibody (PAb) were conjugated
with an appropriate reporter system such as horseradish peroxidase,
fluorescein and colloidal gold. These tagged antibodies may then be
used in antigen-detection immunoassays such as ELISA, histochemical
stain and dipstick test formats. These test formats may be used to
detect parasite antigens in tissues and body fluids of mammalian
hosts and vectors.
[0052] The rabbit anti-leishmania polyclonal antibody preparation
demonstrated high affinity and avidity for the immunogens used to
immunize the rabbits. The PAb produced a very striking Western Blot
pattern similar to that of kala azar patients. See FIG. 7.
[0053] It is appreciated that the present invention also
encompasses monoclonal antibodies against leishmania soluble
antigens, hybridomas producing such, and methods of making and
using thereof. Thus, monoclonal antibodies against leishmania
soluble antigens may be used in the assays described herein. The
monoclonal antibodies may be made by standard methods known in the
art.
EXAMPLE 4
Antibody-Capture Enzyme-Linked Immunosorbent Assay
[0054] Generally, in the solid phase enzyme immunoassay for
Leishmania, soluble antigens of the Leishmania promastigotes were
coated on the inner surface of a test well which serve to bind
specific antibody from a sample. Peroxidase conjugated antibody to
anti-human IgG was added and reacted with bound antibody. A
chromogenic substrate, such as horseradish peroxidase, for
peroxidase was added. If antibody to Leishmania was present, there
was a reaction that resulted in the development of color. Other
fluorescent, chemiluminescent and chromogenic agents may be used
with appropriate enzymes and substrates.
[0055] Plate sensitization was affected by coating a polystyrene,
96-well microtitre plates (Immulon 4, Dynatech Laboratories,
Chantilly, Va.) with 100 .mu.l of the respective exo-antigen
solution (5 .mu.g protein per well). L. donovani (Walter Reed
reference strain 130, clone E) exo-antigen was used to sensitize
plates for visceral and canine leishmaniasis samples, and L.
mexicana (ATCC strain 50157) exo-antigen was used to sensitize
plates for cutaneous leishmaniasis samples. Positive and negative
controls were diluted at the same ratio as the sample. The
dilutions were then placed in the wells of the microtitre plate.
The samples were covered and incubated for 1 hour at room
temperature in a humid environment.
[0056] Each plate was then blocked with 1.0% casein (Sigma Chemical
Co., St. Louis, Mo.) in PBS for one hour at room temperature. The
blocking buffer was removed by aspiration and the serum samples
(100 .mu.l of 1:1000 dilution) and appropriate controls were added
to the microtiter plate and the plate was incubated at 26.degree.
C. for 40 minutes. The plate was washed with 0.05% PBS-Tween-20
(PBS-Tween) buffer four times. Preferably, an automatic plate/strip
washer is used. The well contents were shaken out at the end of the
final wash. Then goat anti-human IgG (whole molecule) conjugated
with horseradish peroxidase (Kirkegaard & Perry Laboratories
Inc., Gaithersburg, Md.) was added at 1:5000 dilution and then the
plate was incubated at 26.degree. C. for an hour in a humid
environment.
[0057] The plate was then washed four times with PBS-Tween buffer
and 100 .mu.l of TMB substrate (KPL. Inc., Gaithersburg, Md.) was
added to each well. The plate was incubated for about 15 minutes in
the dark. The optical density (OD) was periodically read at 650 nm
wavelength in an ELISA plate reader (Molecular Devices, Menlo Park,
Calif.) until the OD value of a reference positive control (S5,
Kala azar patient, Nepal) reached 0.8. At this point 100 .mu.l of a
stop solution (0.1M phosphoric acid) was applied to each well and
the final OD reading was taken immediately at 450 nm. Preferably, a
dual beam ELISA reader is used.
[0058] A reference positive serum was used in all plates, and only
interassay variation of less than 10% was accepted. The lower limit
of positivity (cut off) was determined by the mean of the negative
controls subset+3 standard deviations.
[0059] A. Visceral Leishmaniasis.
[0060] Generally, for the test results to be accepted for visceral
leishmania, the negative control must have an OD reading under 0.15
and the positive control must be over 0.8 at 650 nm. If the
controls do not satisfy this criteria, the test should be repeated.
Samples yielding absorbance values under 0.2 are negative and
samples above 0.2 but below 0.3 may contain antibody but the amount
is lower than the generally accepted significant level. It is noted
that one may designate a more stringent or less stringent range for
determining the absorbance levels that are indicative of exposure
to leishmaniasis antigen. Samples giving absorbency values above
0.3 contain higher levels of antibody that are generally considered
to be at or above the significant level.
[0061] L. donovani WR0130E (ATCC strain 30503) exo-antigen was used
as the material to coat the microtitre plate. The negative control
sera subset gave a negative cutoff score of about 0.225 for the IgG
assay. With respect to specific IgG, all 129 clinically confirmed
positive VL patient sera gave OD readings above the negative cutoff
(100% sensitivity). When measuring IgM, the negative control sera
tested gave a negative cutoff score of about 0.310. The sensitivity
for this assay was 94.57% (122/129 positive).
[0062] B. Cutaneous Leishmaniasis.
[0063] Generally, for the test results to be accepted for cutaneous
leishmaniasis, the negative control must have an OD reading under
0.3 and the positive control must be over 0.8 at 650 nm. If the
controls do not satisfy these criteria, the test should be
repeated. Samples yielding absorbance values under 0.3 are negative
and samples above 0.3 but below 0.3 may contain antibody but the
amount is lower than the generally accepted significant level.
Again it is noted that one may designate a more stringent or less
stringent range for determining the absorbance levels that are
indicative of exposure to leishmaniasis antigen. Samples giving
absorbance values above 0.3 contain higher levels of antibody that
are generally considered to be at or above the significant
level.
[0064] L. mexicana exo-antigen (ATCC strain 50157) was used as the
material to coat the microtitre plate. The negative control sera
subset gave a negative cutoff score of approximately 0.3 for the
IgG assay. The boxplot in FIG. 3 illustrates specific IgG antibody
levels measured in CL patient sera samples. With respect to
specific IgG, 1321143 clinically confirmed positive CL patient sera
gave OD readings above the negative cutoff (92.31% sensitivity).
FIG. 4 is a boxplot depicting specific IgM antibody levels measured
in the same samples. When measuring IgM, the negative control sera
tested gave a negative cutoff score of approximately 0.15. Only a
few samples (n=6) were dramatically above the negative cutoff
score, the majority of values for positive samples at or near the
median value of the negative control subset. This assay failed to
consistently detect specific IgM in CL patient sera samples (37.9%;
22/58 positive). C. Canine Leishmaniasis.
[0065] L. donovani WR0130E (ATCC strain 30503) exo-antigen was the
material used to coat the microtitre plate. The negative control
sera subset gave a negative cutoff score of an OD reading of about
0.1 at 650 nm for the IgG assay. FIG. 5 shows specific IgG antibody
levels measured in canine leishmaniasis sera samples. With respect
to specific IgG, all 41 clinically confirmed positive canine
leishmaniasis sera samples gave OD readings above the negative
cutoff (100% sensitivity). Note the relatively large degree of
separation between the positive and negative control subsets. The
boxplot in FIG. 6 displays specific IgM antibody levels measured in
the same samples. When measuring IgM, the negative control sera
tested gave a negative cutoff score of approximately 0.25. The
sensitivity for this assay was 97.56% (40/41).
[0066] SDS-PAGE and Western Blot analysis confirmed the ELISA
results for both VL and CL patients, canine and all negative
controls. Mini-Protean 11 (Bio-Rad, Hercules, Calif.) was used for
SDS-PAGE. Each antigen preparation was boiled for 5 minutes in
sample buffer without a reducing agent and was immediately
subjected to electrophoresis on 4% stacking, 12.5% separating
bis-acrylamide gels. A wide-range molecular mass marker (Bio-Rad,
Hercules, Calif.) was used. An antigen load of 120 .mu.g of protein
was used in each mini-gel. The gels were run at 100 V of constant
voltage for 1.5 hours in Tris-glycine-SDS buffer (pH 8.3).
[0067] Protein bands from the gel were transferred to
nitrocellulose. Antigens from the SDS-polyacrylamide gels were
electroblotted onto 0.45 .mu.m pore size nitrocellulose membranes
(Bio-Rad, Hercules, Calif.) with standard transfer buffer (0.02 M
Tris, 0.15 M glycine, 0.1% SDS, 20% methanol) and 250 mA of
constant current for 1 hour at 4.degree. C. Following the blotting,
portions of the membranes containing the protein markers were
stained with 0.5% Amido black for 5 minutes and were destained in
distilled water--glacial acetic acid solution. The membranes were
immediately blocked with a 2% skim milk TTBS (100 mM Tris, 0.9%
NaCl, 0.1% Tween 20) solution and kept refrigerated until use.
[0068] Detection of antibodies from subject sera bound to the
antigens of the Western blot was done with an
avidin-biotin-alkaline phosphatase system by methods standard in
the art. Strips 4 mm wide were cut from previously blotted and
blocked membranes. These strips were incubated with diluted sera
(1:3,200 in TTBS) for 30 minutes at room temperature with constant
agitation. After incubation with the primary antibody, the strips
were washed 4 times for 10 minutes each time with TTBS. After the
last wash, biotinylated, anti-human immunoglobulin G was added and
the mixture was incubated for 30 minutes under the same conditions
as described above. Preformed avidin-biotin-alkaline phosphatase
complex was added. The mixture was then incubated under the same
conditions as above. The membranes were developed with a BCIP/NBT
substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.)
for 5 minutes. The reaction was stopped by rinsing the strips with
distilled water and then adding PBS-EDTA (20 mM).
EXAMPLE 5
Antigen-Capture Enzyme-Linked Immunosorbent Assay
[0069] A. General Antigen-Capture ELISA.
[0070] An antigen-capture ELISA ("sandwich" ELISA) format based on
the same soluble antigens and their complementary antibodies was
developed to detect active infection in vertebrate hosts and sand
fly vectors.
[0071] The capture polyclonal antibody was adsorbed to the wells of
a microtiter plate. After the capture polyclonal antibodies were
bound to the plate, the well contents were aspirated and the
remaining active binding sites on the plates were blocked with
blocking buffer. A sample such as patient sera, urine, or ground
sand flies was then tested. Test samples were appropriately diluted
with blocking buffer and an aliquot was tested. Sand flies to be
tested were ground in blocking buffer with Nonidet P-40 (ELISA
grade Sigma casein, bovine milk) and an aliquot was tested.
Positive and negative controls were also added.
[0072] If parasite antigen was present it formed antigen-antibody
complexes with the polyclonal sera used to coat the plate. After a
2-hour incubation, the sample was aspirated and the wells were
washed. The peroxidase-linked polyclonal sera were then added to
the wells, thereby completing the formation of the sandwich. Other
chromogenic agents such as colloidal gold and FITC may be used with
their corresponding substrates.
[0073] After 1 hour, the well contents were aspirated, the plate
was washed and a clear peroxidase substrate solution from
Kirkegaard & Perry Laboratories (Gaithersburg, Md.) was added.
As the peroxidase enzyme reacted with the substrate a dark product
was formed, which the intensity of its color was relative to the
amount of circulating antigen present in the test sample.
Quantitative results were obtained by making an endpoint
determination a few minutes after the substrate has been added by
measuring the optical density of the well contents at 450 nm with
an ELSIA plate reader. However, qualitative results may be read
visually in the field. ELISA positive samples may be retested to
confirm positives and to estimate the amount of circulating antigen
per sample.
[0074] Recently, the antigen-specific, conjugated polyclonal
antibodies were used in combination with unconjugated PAb and
monoclonal antibodies (MAb) specific for leishmanial Secretory Acid
Phosphatases (S-cAcP) to develop a simple antigen-capture assay.
The sensitivity of this assay was tested with serial dilutions of
the antigen preparation prepared from the method explained above.
Specific activity was recorded with the use of an HRP conjugate at
a 1:32,000 dilution. The level of sensitivity in measuring these
antigens with PAb was 400 ng/ml.
[0075] B. Rapid Wicking Assay.
[0076] A rapid wicking assay, based on the dual "sandwich" ELISA,
was developed. The assay is conducted by placing a dipstick
impregnated with immobilized polyclonal antibodies from rabbits
immunized with the soluble antigen (Cellabs Pty, Ltd. Sydney,
Australia) into a test solution. When soluble leishmanial antigen
is present in the solution, it binds to a specific antibody with a
gold sol particle label. As the antigen-antibody-gold complexes
migrate through a test zone on the dipstick comprising immobilized
polyclonal antibodies from rabbits immunized with the soluble
antigen (Cellabs Pty, Ltd. Sydney, Australia), they bind to the
corresponding immobilized antibodies to form a "sandwich". The
unbound dye complexes migrate out of the test zone and can be
captured later in a control zone. A reddish-purple line develops in
the specific area of the test zone when antigen is present. A
control line in the control zone should develop provided that the
test was conducted correctly.
[0077] The test zone comprises immobilized polyclonal antibodies
from rabbits immunized with the soluble antigen (Cellabs Pty, Ltd.
Sydney, Australia). The control zone comprises antibodies to
immobilized polyclonal antibodies from rabbits immunized with the
soluble antigen (Cellabs Pty, Ltd. Sydney, Australia). Monoclonal
antibodies against the soluble antigen may be used.
[0078] The test samples may be sand flies or other organisms
comprising leishmania parasites ground up in a solution such as PBS
with 0.5% casein. Alternatively the test samples may be blood,
serum, urine, mucus, tears, stool or the like, obtained from a
subject. When the test sample is urine, it is preferably
undiluted.
EXAMPLE 6
In vitro Direct Immunofluorescence Test
[0079] Fluorescein-labeled polyclonal antibodies raised against
specific antigens of Leishmania parasites may be used in an in
vitro direct immunofluorescence assay. The labeled antibody binds
specifically to the antigens present on the surface of the parasite
which can be detected in a variety of smears such as vector
specimen, in vitro culture material and patient biopsy smears.
[0080] Smears were prepared on glass slides and fixed with
methanol. Unbound antibodies were removed by washing. When viewed
under a fluorescence microscope, Leishmania parasites were seen as
bright apple-green organisms characteristic to their life cycle
stages contrasted with the reddish brown color of counterstained
material. Promastigotes in the vectors or in culture were detected
by their characteristic long and slender body (about 20 .mu.m in
length) with an anterior flagellum. Amastigotes present in clinical
samples were detected by their characteristic round or oval shape
measuring about 2-5 .mu.m in diameter.
[0081] Generally, sample smears were prepared on slides marked with
wells or on plain glass slides. Suitable smears had adequate
specimen and were moderately thin. For sample smears prepared on a
single well slide, an adequate amount of fluorescein-labeled
purified polyclonal antibody diluted in a protein stabilized buffer
solution (pH 7.4) with Evans Blue as a counter stain and 0.1% w/v
sodium azide was added to the fixed sample smear and positive
control.
[0082] For smears prepared on plain glass slides, after drying, the
smear was pretreated by dipping the slide in a Coplin jar
containing 0.1% sodium deoxycholate prepared in 0.85% NaCL for 5-10
minutes. The smear was air-dried. Then an adequate amount of
fluorescein-labeled purified polyclonal antibody diluted in a
protein stabilized buffer solution (pH 7.4) with Evans Blue as a
counter stain and 0.1% w/v sodium azide was added to the fixed
sample smear and positive control.
[0083] The slides were incubated at 37.degree. C. in a moist
chamber for 30 minutes in the dark. Then the slides were rinsed in
a saline bath for about 2-5 minutes. The slides were allowed to air
dry and then mounted with coverslips. With a fluorescence
microscope under oil immersion, the slides were read.
[0084] This test format was found to be very sensitive to detecting
amastigotes in infected patient tissues from subjects with
cutaneous leishmaniasis infected with L. brasiliensis and splenic
aspirates from subject with visceral leishmaniasis infected with L.
donovani. It may be used to highlight surface antigens and cellular
structure in cultured promastigotes. Preliminary results from gene
cloning experiments with the L. donovani clone used to produce the
exo-antigen indicate that one of the major immunogens in the
sensitization of the rabbits used to generate the PAb is a major
surface antigen.
EXAMPLE 7
Competitive ELISA
[0085] Microtiter plates were coated with the anti-leishmania PAb
and blocked with 1% yeast extract. The exo-antigen was labeled with
HRP. The resulting exo-antigen conjugate was mixed 1:1 with samples
and applied to the plate and incubated overnight at 4.degree. C.
The plate was washed 3 times with PBS and a substrate, ABTS, was
applied. The optical density of the samples were read at 405
nm.
[0086] The competitive ELISA format worked to indirectly detect
free antigen in samples. There was a correlation between increasing
amounts of antigen in the samples and reduced optical density
values. Further experiments were conducted to demonstrate that this
relationship remained intact when varying the amount of the labeled
or unlabeled antigen in the test sample and to demonstrate the
importance of coating and blocking wells prior to sample
incubations. See FIGS. 8 and 9.
EXAMPLE 8
Assay Optimization
[0087] The antibody detection ELISA may be optimized for diagnosis
of visceral and cutaneous leishmaniasis. For example, for assay
optimization for VL, the antigens released by L. donovani
promastigotes (WHO Reference Strain 065) in vitro at 26.degree. C.
for 72 hours into serum-free and protein-free medium were obtained
as described in Martin et al., (1998) and used. The soluble
antigens were separated by SDS-PAGE using 4-15% Novex gradient gels
(Novex, San Diego, Calif.). The proteins were visualized by
colloidal Coomassie blue G-250 and silverstaining. The molecular
weights of the secreted antigens were estimated with the reference
to a prestained standard (Novex, San Diego, Calif.).
[0088] Titration and checker-board analyses were performed to
optimize the assay protocol. Optimal results were obtained when
antigen (50 .mu.g/ml) was coated with PBS-methyl glyoxal buffer and
the wells were blocked with 0.5% casein. It was found that a serum
dilution of 1:500 in antigen-coated wells blocked with 0.5% casein
generated lowest absorbance with negative control sera and higher
absorbance with positive sera, sera from well-characterized,
culture positive case subjects (used as reference positives).
[0089] An equal number of sera from North American naives with no
travel history to leishmania endemic areas were used as reference
negatives, negative controls or negative sera. These sera samples
were used as reference samples to optimize the assay. The reference
sera were not pooled and were used as individual data points. After
optimizing the assay, individual sera samples obtained from endemic
areas of N. Africa were screened and used to show assay performance
in terms of specificity and sensitivity.
[0090] Two ELISA plates, Greiner (Greiner Labortecnik ELISA plate
Cat # 705071) and Labsystem (Labsystem combiplate breakable 8 Cat
#95029400), were evaluated.
[0091] The following four different coating buffers (a) 0.2M
carbonate/bicarbonate buffer pH 9.6, (b) 0.01M phosphate buffer
(PO.sub.4) pH 7.2, (c) blank culture medium (serum-free) pH 7.2,
and (d) 0.1M phosphate buffered saline (pH 7.2)+1% methyl glyoxal
were tested for their ability to immobilize the antigen onto the
solid phase ELISA wells.
[0092] Gelatin ranging in concentration from 0.1 to 0.4% solution
and 0.5% casein were tested. BSA and other routinely employed
blocking agents were not tested because of non-specific binding
noted in previous experiments.
[0093] PBS/T and PBS/T+0.2M NaCl were used as the washing buffers.
An automatic plate washer (Denley Well Wash 04) with 4 cycle wash
in each step was used to wash the ELISA wells. In most of the
assays the wash buffer was used as the diluent for sera as well as
the conjugated detector antibody.
[0094] Two anti-human IgG conjugated to HRP (HRP conjugate) were
tested. A goat anti-human IgG-HRP (Kirkegaard & Perry
Laboratories, Gaithersburg, Md.), a polyclonal conjugate (PAb
conjugate), and a mouse anti-human IgG-HRP (Cellabs, Brookvale,
Australia), (MAb conjugate).
[0095] All steps of the ELISA were performed at room temperature.
The S/N ratio, the differential absorbance between the negative
sample versus the test or positive sera sample, was used to
quantify the specific antigen and antibody reactions. An anti-human
monoclonal antibody conjugated with HRP (MAb conjugate)
outperformed a commercially available anti-human polyclonal
antibody conjugate (PAb conjugate) (Cellabs Pty, Ltd. Sidney,
Australia). The MAb conjugate gave minimal background reactions
with endemic sera. Generally, the wells of an ELISA plate were
coated with 50 .mu.g/mL of the soluble antigens released by L.
donovani promastigotes in serum-free and protein-free medium mixed
in PBS-methyl-glyoxal overnight. After removing the antigen, the
wells were blocked with 0.5% casein for 1 hour at room temperature.
Test sera along with positive and negative control sera diluted
1:500 in PBS/T, were reacted for 1 hour at room temperature. After
washing the plate with PBS/T, the wells were reacted with an
HRP-conjugated anti-human antibody, the detector antibody, for 40
minutes at room temperature. The plates were washed and specific
binding of antigens on the solid phase and the specific antibodies
present in the test sera were measured indirectly by the binding of
HRP labeled detector antibody which was further detected by using
TMB+H.sub.2O.sub.2 as a chromogenic substrate.
[0096] TMB solution A and B (KPL, Gaithersburg, Md.) were mixed in
equal parts 5-10 minutes before and transferred an aliquot of 100
.mu.l to each well as per the guidelines provided by the
manufacturer. During the developmental phase of the assay, the
color intensity of positive sera wells was monitored (absorbance OD
of 620 nm) and stop solution was added when those wells reached an
OD of 0.450. In the final optimized assay, the incubation time with
substrate was fixed at 25 minutes.
[0097] After adding the stop solution (1M Phosphoric acid prepared
in distilled water) the contents were mixed and the plate was read
at dual filter (450/620 nm) using a plate reader (Anthos LabTec
Instruments 2001).
[0098] The raw data from the plate, i.e., absorbance at 450/620 nm,
were plotted into histograms and graphs. The relative specific
binding was quantified by the signal to noise (S/N) ratio which was
calculated by dividing the mean absorbance, i.e., absorbance at
450/620 nm, of test sera with the mean absorbance of negative sera,
and plotted. The S/N ratio was directly proportional to the
specific antibody reactivity in ELISA. A batch of n=22 endemic sera
from North Africa were evaluated and resulted with 100% specificity
and sensitivity, 99.99% PPV (positive predictive values) and 95.45%
NPV (negative predictive values).
[0099] The LabSystem plate did not perform well in this study.
There was no discrimination between the positive and negative sera.
There was a clear distinction between sera in the Greiner plate. It
was found that the PBS+methyl glyoxal, phosphate buffer and culture
medium respectively showed higher reactivity with positive sera and
relatively less reactivity with negative sera. A higher S/N ratio
was seen using sera at a 1:500 sera dilution. Phosphate buffer,
culture medium and PBS-glyoxal gave higher SIN ratios. Therefore,
the Greiner plate and PBS+methyl glyoxal were selected as ELISA
plate and coating buffer respectively.
[0100] Using the Greiner ELISA plate and PBS+methyl glyoxal as the
coating buffer for the test, positive and negative sera were used
at 1:500 dilution. Two anti-human IgG-HRP conjugates were evaluated
at 4 different dilutions. The PAb conjugate was found to be highly
reactive with the negative sera indicating a high level of
nonspecific reaction. On the other hand, the same level of
reactivity was observed in the blank and negative sera sample wells
with the MAb conjugate. The reactivity with MAb conjugate appeared
to be more specific with positive sera as evidenced by higher S/N
ratios.
[0101] Negative and positive sera were reacted at 1:500 dilution
and washed with two different wash solutions. MAb conjugate was
used at 1:8000 dilution. Plates were read at 450/620 nm. Reactivity
of blanks and negative sera were lower in wells washed with PBS/T
+0.2M NaCl. The data indicated that PBS/T +0.2M NaCl was more
effective in removing non-specific binding (nearly 45% reduction of
nonspecific signal 0.283 v. 0.114) and increasing the SIN ratio
from 3.7 to 7.2. Despite its higher S/N ratio, 0.2M NaCl was left
out of the final wash buffer because it formed a precipitate on
standing.
[0102] Having selected the Greiner plate and PBS+glyoxal as the
coating buffer, optimum levels of antigen and appropriate blocking
reagents were investigated. Wells were coated with a series of
antigen concentration from 1.25 .mu.g/ml to 40 .mu.g/ml. Two
blocking reagents, 0.4% gelatin and 0.5% casein prepared in
distilled water were evaluated. Positive and negative sera, diluted
1:500 in PBS/T, reacted in the Ag-coated and blocked wells for 1
hour at room temperature. Two conjugates, PAb and MAb-conjugates,
diluted 1:4000 in PBS/T were added to wells and incubated for 30
minutes at room temperature. The color was developed for 25 minutes
by adding the substrate and immediately read after addition of
stopping solution. The reactivity was higher in blank and negative
sera wells with PAb conjugate thereby reducing the differences
between samples that resulted with a low S/N ratio. With the MAb
conjugate, the absorbance of blank and negative sera wells was
almost equal. There was a pattern in the reactivity relative to the
antigen concentration. Wells reacted with the positive sera showed
a gradual rise in absorbance dependent upon the antigen
concentration. On the whole, MAb conjugate reactivity was
relatively lower in control sample wells. The SIN ratios were
higher with MAb conjugate when casein used for blocking. MAb
conjugate with casein blocking generated excellent SIN ratios, at
20 and 40 .mu.g/ml antigen levels.
[0103] Experiments involving the relative kinetics of antibody
reactivity at different sera dilutions provided a good
discrimination at 1:500 dilution. This formed the basis for future
assays.
[0104] After optimizing assay steps, the following protocol was
followed for evaluating test sera samples of subjects from endemic
areas. In short, the wells were coated with 50 .mu.g/ml soluble
antigen mixed in PBS-methyl-glyoxal buffer overnight and after
removing the antigen, the wells were blocked with 0.5% casein for 1
hour at room temperature. Test serum along with control sera
diluted 1:500 in PBS/T, was reacted for 1 hour at room temperature.
After washing the plate in PBS/T, the wells were reacted with MAb
conjugate at 1:8000 dilution for 40 minutes at room temperature and
after washing, the TMB substrate was added and color development
was allowed to proceed for 25 min and then stopped with the stop
solution.
[0105] Absorbance was read at dual filter (4501620 nm) and the
results were analyzed. A total of n=22 test clinical sera obtained
from the endemic areas of North Africa were evaluated along with
n=5 reference control negative sera.
[0106] SDS-PAGE analysis was conducted Coomassie staining showed
several major bands with approximate molecular weights of 11, 30,
42, 50 and 161 kDa. In addition to these abundant bands, silver
staining revealed more distinct protein bands of approximately 6,
15; 17, 22, 58, and 107 kDa. This illustrates that the test
contained a variety of protein antigens.
[0107] The cut-off value in the current assay was mean +3 SD of
negative (n=5) sera (Mean 0.1304, SD=0.042), i.e., 0.278 which is
rounded off to 0.300. Using an absorbance OD450/620 nm of 0.300 as
the cut off, n=22 test sera from field were categorized as either
positive or negative. With the exception of one sample, all were
positive. The sensitivity, specificity, PPV and NPV were
calculated. Thus, the sensitivity and specificity were both 100%,
the PPV was 99.9% and NPV was 95.45%. The resultant S/N ratio of
these samples suggests that the assay is highly sensitive and
specific.
[0108] Clearly, one of ordinary skill in the art may further
optimize the assays of the invention by changing various assay
conditions by methods standard in the art.
[0109] Incorporation by Reference
[0110] To the extent necessary to understand or complete the
disclosure of the present invention, all publications, patents, and
patent applications mentioned herein are expressly incorporated by
reference therein to the same extent as though each were
individually so incorporated.
* * * * *